Pyridinium boronic acid quenchers for use in analyte sensors

ABSTRACT

Novel pyridinium salts functionalized with boronic acid and methods of making them are disclosed. When combined with a fluorescent dye, the compounds are useful in the detection of polyhydroxyl-substituted organic molecules.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/915,372 filed May 1, 2007, which is hereby expressly incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to the detection ofpolyhydroxyl-substituted organic molecules, and in particular to the useof pyridinium salts functionalized with boronic acids as quenchers offluorescent dyes.

DESCRIPTION OF THE RELATED ART

Investigators have made fluorophores with N-benzyl-2-boronic acidpyridinium groups attached to a porphyrin ring (Arimori, S. et al. 1996J Am Chem Soc 118:245-246). They were used to promote aggregation withanother porphyrin substiuted with saccharide groups via intermolecularester formation. Benzyl-2- and benzyl-4-boronic acid substituents on thepyridine nitrogens in substituted porphyrins were also described(Arimori et al. 1996 Chemistry Letters 25:77). They were used todistinguish chiral orientation in sugars. It was shown that thefluorescence was reduced by complex formation between these porphyrinsand anthraquinone disulfonates. The complex was dissociated by reactionof the boronic acids with fructose resulting in an increase influorescence. The quenching moiety in this case was the anthraquinonecomponent (Arimori et al. 1995 J Chem Soc, Chem Commun 9:961-962).Subsequently, investigators described a dye with a pyridine ring in thestructure, substituted on the nitrogen with a benzyl-2-boronic acidgroup (Takeuchi et al, 1996 Bull Chem Soc (Jpn) 69:2613-2618). It wasnoted that the pyridinium group in ortho-position enhances reactivity ofboronic acids with diols. This dye was used to detect nucleotides. In apaper concerning trialkyl ammonium substituted benzyl-2-boronic acids, ageneric formula for N-benzyl-2-boronic acid derivatives ofpara-substituted pyridines was given, where the substituent wasspecified as an R-group (i.e., alkyl) (Takeuchi et al. 1996 Tetrahedron52:12931-12940).

A pyridinium salt without a boronic acid substituent was used as areference compound in a quenching study (Cordes et al. 2005 Langmuir21:6540-6547). Other investigators measured the fluorescence quenchingactivity and glucose response of the three isomers of N-benzylboronicacid pyridinium salts. These compounds showed poor quenching of pyraninefluorescence and gave no glucose response (See e.g., “Detection ofglucose with arylboronic acid containing viologens and fluorescent dyes:Progress toward a continuous glucose monitoring system for in vivoapplications” Cappuccio, Frank E., Ph.D. Dissertation; UNIVERSITY OFCALIFORNIA, SANTA CRUZ, 2004).

A comparative study was reported on the quenching of Ru(bpy)₃ by methylviologen (MV) and a series of 4-substituted N-methyl pyridiniums (Jonesand Malba 1985 J Org Chem 50:5776-5782). This study showed thatpyridiniums substituted in the 4-position with electron withdrawinggroups conjugated to the ring behaved like MV. These compounds showedreversible reduction at similar potentials and had Stern-Volmer (S-V)constants in the same range.

Alkyl pyridinium surfactants have been widely studied as fluorescencequenchers. Fluorophores that have been successfully quenched includepolycyclic aromatic hydrocarbons (Pandey et al. 1999 Talanta48:1103-1110; Palit et al. 1997 Chem Phys Lett 269:286-292; Wadek andTucker 2000 Talanta 53:571-578; Mao et al. 2003 J Sep Sci 26:1643-1649),aminofluorene (Saha et al. 1999 J Photochem Photobiol A 121:191-198),and carbazole substituents on polymers (Yatsue et al. 1992 J Phys Chem96:10125-10129).

Most studies were with simple N-alkyl pyridinium salts where the alkylgroup was large enough to make the salt surface active. The polymerstudy was carried out with para-substituted N-alkyl pyridiniums,including derivatives of 4-acetyl pyridine, methyl isonicotinate, andisonicotinamide. In other studies with ring substituted pyridiniums,bis-picolinium salts with N,N′-alkylene bridging groups were used toquench the fluorescence of naphthols. The quenching efficiency of thebis compounds was substantially higher than that of a mono-picoliniumcontrol; and was highest for the compound with a methylene linker.(Panda et al. 1998 J Photochem Photobio A 113:73-80).

SUMMARY OF THE INVENTION

A terpyridinium quencher having the structure (T-1) below is disclosedin accordance with preferred embodiments of the present invention.

A method of making T-1 is disclosed in accordance with anotherembodiment of the present invention comprising the steps of:

A terpyridinium quencher having the structure (T-2) below is disclosedin accordance with preferred embodiments of the present invention.

A method of making T-2 is disclosed in accordance with anotherembodiment of the present invention comprising the steps of:

Pyridinium quenchers having generic structures as shown below aredisclosed in accordance with preferred embodiments of the presentinvention.

A. Reactive Compound:

wherein:

-   -   X— is a counterion;    -   X¹ is —O— or —NH—;    -   X² is —O— or —NH—;    -   L is a divalent linking group selected from a direct bond or, a        lower alkylene having 1 to 8 carbon atoms, optionally terminated        with or interrupted by one or more divalent connecting groups        selected from sulfonamide (—SO₂NH—), amide —(C═O)N—, ester        —(C═O)—O—, ether —O—, sulfide —S—, sulfone (—SO₂—), phenylene        —C₆H₄—, urethane —NH(C═O)—O—, urea —NH(C═O)NH—, thiourea        —NH(C═S)—NH—, amide —(C═O)NH—, amine —NR— (where R is defined as        alkyl having 1 to 6 carbon atoms);    -   Z is either a polymerizable ethylenically unsaturated group        selected from but not limited to methacrylamido-, acrylamido-,        methacryloyl-, acryloyl-, or styryl- or optionally Z is a        reactive functional group, capable of forming a covalent bond        with a polymer or matrix. Such groups include but are not        limited to —Br, —OH, —SH, —CO₂H, and —NH₂. In one embodiment, Z        is

-   -    wherein R is H or CH₃;

is substituted on the pyridinium ring in the meta or para position; and

-   -   —B(OH)₂ may be in the ortho, meta or para position.        B. Non-Reactive Compound:

wherein

-   -   X is —O— or —NH—; and    -   R′ is an alkyl, optionally including —O— units in the carbon        chain and terminated with —OH or —OCH₃.

A pyridinium quencher having the structure (P-1) below is disclosed inaccordance with preferred embodiments of the present invention.

A method of making P-1 is disclosed in accordance with anotherembodiment of the present invention comprising the steps of:

A pyridinium quencher having the generic structure below is disclosed inaccordance with preferred embodiments of the present invention.

wherein

-   -   Z is a reactive, ethylenically unsaturated group selected from        but not limited to methacrylamido-, acrylamido-, methacryloyl-,        acryloyl-, or styryl- or optionally Z is a reactive functional        group, capable of forming a covalent bond with a polymer or        matrix. Such groups include but are not limited to —Br, —OH,        —SH, —CO₂H, and —NH₂. In one embodiment, Z is

-   -    wherein R is H or CH₃;    -   Y is a trivalent connecting group selected from

-   -    where R is H or a lower alkyl, and

-   -   X¹ and X² are —O— or —NH—; and    -   L¹, L², and L³ are selected from a direct bond or, a lower        alkylene having 1 to 8 carbon atoms, optionally terminated with        or interrupted by one or more divalent connecting groups        selected from sulfonamide (—SO₂NH—), amide —(C═O)N—, ester        —(C═O)—O—, ether —O—, sulfide —S—, sulfone (—SO₂—), phenylene        —C₆H₄—, urethane —NH(C═O)—O—, urea —NH(C═O)NH—, thiourea        —NH(C═S)—NH—, amide —(C═O)NH—, amine —NR— (where R is defined as        alkyl having 1 to 6 carbon atoms) or combinations thereof.

A pyridinium quencher having the structure (P-2) below is disclosed inaccordance with preferred embodiments of the present invention.

wherein n=1-10.

A method of making P-2 is disclosed in accordance with anotherembodiment of the present invention comprising the steps of:

Another pyridinium boronic acid quencher having the generic structurebelow is disclosed in accordance with preferred embodiments of thepresent invention.

wherein

-   -   X⁻ is a counterion;    -   X¹ is —O— or —NH—;    -   X² is —O— or —NH—;    -   L is a divalent linking selected from a direct bond or, a lower        alkylene having 1 to 8 carbon atoms, optionally terminated with        or interrupted by one or more divalent connecting groups        selected from sulfonamide (—SO₂NH—), amide —(C═O)N—, ester        —(C═O)—O—, ether —O—, sulfide —S—, sulfone (—SO₂—), phenylene        —C₆H₄—, urethane —NH(C═O)—O—, urea —NH(C═O)NH—, thiourea        —NH(C═S)—NH—, amide —(C═O)NH—, amine —NR— (where R is defined as        alkyl having 1 to 6 carbon atoms) or combinations thereof;    -   Z is a reactive group selected from a coupling group or an        olefinically unsaturated group, or Z is

-   -    wherein R is H or CH₃;    -   the bond from the central benzene ring is to the ortho, meta or        para position on the adjacent pyridinium rings; and    -   —B(OH)₂ may be in the ortho, meta or para position.

A specific pyridinium boronic acid quencher, termed P-3, having thestructure below is disclosed in accordance with preferred embodiments ofthe present invention.

A method of making P-3 is disclosed in accordance with anotherembodiment of the present invention comprising the steps of:

Another pyridinium boronic acid quencher having the generic structurebelow is disclosed in accordance with preferred embodiments of thepresent invention.

wherein

-   -   X⁻ is a counterion;    -   X¹ is —O— or —NH—;    -   X² is —O— or —NH—;    -   L is a divalent linking selected from a direct bond or, a lower        alkylene having 1 to 8 carbon atoms, optionally terminated with        or interrupted by one or more divalent connecting groups        selected from sulfonamide (—SO₂NH—), amide —(C═O)N—, ester        —(C═O)—O—, ether —O—, sulfide —S—, sulfone (—SO₂—), phenylene        —C₆H₄—, urethane —NH(C═O)—O—, urea —NH(C═O)NH—, thiourea        —NH(C═S)—NH—, amide —(C═O)NH—, amine —NR— (where R is defined as        alkyl having 1 to 6 carbon atoms) or combinations thereof;    -   Z is either a polymerizable ethylenically unsaturated group        selected from but not limited to methacrylamido-, acrylamido-,        methacryloyl-, acryloyl-, or styryl- or optionally Z is a        reactive functional group, capable of forming a covalent bond        with a polymer or matrix. Such groups include but are not        limited to —Br, —OH, —SH, —CO₂H, and —NH₂. In one embodiment, Z        is

-   -    wherein R is H or CH₃;    -   the ambiguously depicted bonds are in the ortho, meta or para        position; and    -   —B(OH)₂ may be in the ortho, meta or para position.

Another pyridinium boronic acid quencher, termed P-4, having thestructure below is disclosed in accordance with preferred embodiments ofthe present invention.

A method of making P-4 is disclosed in accordance with anotherembodiment of the present invention comprising the steps of:

In one embodiment, an analyte sensor is disclosed comprising afluorophore configured to absorb light at a first wavelength and emitlight at a second wavelength and a quencher configured to modify thelight emitted by the fluorophore by an amount related to the analyteconcentration, wherein the quencher comprises a boronic acid-substitutedpyridinium. In preferred embodiments, the quencher is selected from thegroup consisting of P1, P2, P3 and P4.

A glucose sensor is disclosed in accordance with another embodiment ofthe present invention, comprising any one or more analyte-bindingmoieties selected from the group consisting of T-1, T-2, P-1, P-2, P-3and P-4; a fluorescent dye, e.g., HPTS-triCys-MA; and optionally ananalyte permeable component, e.g., a polymer matrix or a semipermeablemembrane, that provides a means for immobilizing the dye and quencher.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the glucose response of a sensor comprising quencherP-1 and dye HPTS-triCys-MA immobilized within a hydrogel at the tip ofan optical fiber. The detection chemistry was excited at 470 nm andfluorescence was measured between 520-700 nm in the presence ofincreasing concentrations of glucose.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Fluorescent dyes and analyte-binding moieties that modulate fluorescenceupon binding analyte are known and have been used in indicator systemsfor analyte detection. See e.g., U.S. Pat. Nos. 6,653,141, 6,627,177,5,512,246, 5,137,833, 6,800,451, 6,794,195, 6,804,544, 6,002,954,6,319,540, 6,766,183, 5,503,770, and 5,763,238; and co-pending U.S.patent application Ser. Nos. 10/456,895, 11/296,898, 11/671,880,60/833,081, 60/888,477, and 60/888,475; each of which is incorporatedherein in its entirety by reference thereto.

Although Applicants do not intend to be bound by the proposed mechanismof action, one mechanism that may be employed in some of the preferredindicator systems described in the above-referenced co-pending U.S.patent applications includes inter alia the formation of a ground statecomplex between the analyte-binding moiety and the fluorescent dye. As aresult of the formation of the complex, the fluorescence may bequenched. When the boronic acid group on the preferred analyte-bindingmoiety reacts with a polyhydroxyl-substituted organic molecule such asglucose, the boron becomes negatively charged. This weakens the complex,resulting in dissociation, and an increase in fluorescence that isrelated to glucose concentration.

The indicator systems of the present invention for the detection ofpolyhydroxyl-substituted organic molecules (e.g., sugars) comprise anovel class of pyridinium salts functionalized with boronic acids as theanalyte binding moieties. In embodiments of the present invention, theanalyte-binding pyridinium quenchers exhibit one or more of thefollowing characteristics. They are: 1) compatible with aqueous media;2) substituted with boronic acid groups; 3) inherently positivelycharged, preferably with at least one cationic group per boronic acid;and 4) amenable to immobilization. The analyte-binding quenchers arehypothesized to be good electron acceptors and the electron transferprocess is reversible.

As used herein, “boronic acid” refers to a structure —B(OH)₂. It isrecognized by those skilled in the art that a boronic acid may bepresent as a boronate ester at various stages in the synthesis of thequenchers of this invention. Boronic acid is meant to include suchesters.

“Fluorophore” refers to a substance that when illuminated by light at aparticular wavelength emits light at a longer wavelength; i.e., itfluoresces. Fluorophores include organic dyes, organometallic compounds,metal chelates, fluorescent conjugated polymers, quantum dots ornanoparticles and combinations of the above. Fluorophores may bediscrete moieties or substituents attached to a polymer. “Fluorescentdye” or “dye” is selected from a discrete compound or a reactiveintermediate which is convertible to a second discrete compound, or to apolymerizable compound.

“Linking group”, also termed “L”, refers to divalent moieties thatcovalently connect the sensing moiety to the polymer or matrix. Examplesof L include those which are each independently selected from a directbond or, a lower alkylene having 1 to 8 carbon atoms, optionallyterminated with or interrupted by one or more divalent connecting groupsselected from sulfonamide (—SO₂NH—), amide —(C═O)N—, ester —(C═O)—O—,ether —O—, sulfide —S—, sulfone (—SO₂—), phenylene —C₆H₄—, urethane—NH(C═O)—O—, urea —NH(C═O)NH—, thiourea —NH(C═S)—NH—, amide —(C═O)NH—,amine —NR— (where R is defined as alkyl having 1 to 6 carbon atoms) andthe like.

“Quencher” refers to a compound that reduces the emission of afluorophore when in its presence.

“Pyridinium” refers to a pyridine substituted on the nitrogen to form apositively charged onium salt, optionally substituted at other positionson the pyridine ring.

Pyridinium Quenchers

Pyridinium salts functionalized with boronic acids for use asanalyte-binding quenchers have been synthesized. In accordance withpreferred embodiments, useful pyridiniums are substituted with carbonylgroups and are structurally configured and functionally adapted to bindpolyhydroxyl-substituted organic molecules (e.g., sugars) and may beused in chemical indicator systems of glucose sensors as alternatives toviologen-boronic acid adducts, such as 3,3′-oBBV or derivatives thereof,described e.g., in co-pending U.S. patent application Ser. Nos.11/296,898 and 11/671,880.

The moiety that provides recognition of polyhydroxyl-substituted organicmolecules (e.g., glucose) is an aromatic boronic acid. The boronic acidis either attached via a linker group or covalently bonded to aconjugated nitrogen-containing heterocyclic aromatic structure. Theboronic acid substituted quencher preferably has a pKa of between about4 and 9, and reacts reversibly with glucose in aqueous media at a pHfrom about 6.8 to 7.8 to form boronate esters. The extent of reaction isrelated to glucose concentration in the medium. Formation of a boronateester diminishes quenching of the fluorphore by the pyridinium resultingin an increase in fluorescence dependent on glucose concentration.

A generic structure of a reactive compound is shown below:

wherein:

-   -   X— is a counterion;    -   X¹ is —O— or —NH—;    -   X² is —O— or —NH—;    -   L is a divalent linking selected from a direct bond or, a lower        alkylene having 1 to 8 carbon atoms, optionally terminated with        or interrupted by one or more divalent connecting groups        selected from sulfonamide (—SO₂NH—), amide —(C═O)N—, ester        —(C═O)—O—, ether —O—, sulfide —S—, sulfone (—SO₂—), phenylene        —C₆H₄—, urethane —NH(C═O)—O—, urea —NH(C═O)NH—, thiourea        —NH(C═S)—NH—, amide —(C═O)NH—, amine —NR— (where R is defined as        alkyl having 1 to 6 carbon atoms) or combinations thereof;    -   Z is either a polymerizable ethylenically unsaturated group        selected from but not limited to methacrylamido-, acrylamido-,        methacryloyl-, acryloyl-, or styryl- or optionally Z is a        reactive functional group, capable of forming a covalent bond        with a polymer or matrix. Such groups include but are not        limited to —Br, —OH, —SH, —CO₂H, and —NH₂. In one embodiment, Z        is

-   -    wherein R is H or CH₃;

is substituted on the pyridinium ring in the meta or para position, and

-   -   —B(OH)₂ may be in the ortho, meta or para position.

A generic structure of a non-reactive compound is shown below

wherein

-   -   X is —O— or —NH—; and    -   R′ is an alkyl, optionally including —O— units in the carbon        chain and terminated with —OH or —OCH₃.

A pyridinium quencher having the structure (P-1) below is disclosed inaccordance with preferred embodiments of the present invention.

A method of making P-1 is disclosed in accordance with anotherembodiment of the present invention comprising the steps of:

A pyridinium quencher having the generic structure below is disclosed inaccordance with preferred embodiments of the present invention.

wherein

-   -   Z is a reactive group as previously defined;    -   Y is a trivalent connecting group selected from

-   -    where R is H or a lower alkyl, and

-   -   X¹ and X² are —O— or —NH—; and    -   L¹, L², and L³ are selected from a direct bond or, a lower        alkylene having 1 to 8 carbon atoms, optionally terminated with        or interrupted by one or more divalent connecting groups        selected from sulfonamide (—SO₂NH—), amide —(C═O)N—, ester        —(C═O)—O—, ether —O—, sulfide —S—, sulfone (—SO₂—), phenylene        —C₆H₄—, urethane —NH(C═O)—O—, urea —NH(C═O)NH—, thiourea        —NH(C═S)—NH—, amide —(C═O)NH—, amine —NR— (where R is defined as        alkyl having 1 to 6 carbon atoms) or combinations thereof.

A pyridinium quencher having the structure (P-2) below is disclosed inaccordance with preferred embodiments of the present invention.

wherein n=1-10.

One purpose of the bridging group between the boronic acidfunctionalized pyridinium groups is to allow the two boronic acids tobind cooperatively to one glucose molecule. The inventors hypothesizethat this may result in enhanced glucose selectivity. Rather than beinga simple carbon chain (as illustrated, wherein n=1-10), the bridginggroup could also include other chemical linkages, such as e.g., ethyleneoxide segments. P-2 is representative of a family of poly benzylboronicacid pyridinium salts wherein the pyridinium rings are connected by abridging group attached at the meta- and para-positions through acarbonyl substituent.

A method of making P-2 is disclosed in accordance with anotherembodiment of the present invention comprising the steps of:

Another pyridinium boronic acid quencher having the generic structurebelow is disclosed in accordance with preferred embodiments of thepresent invention.

wherein

-   -   X⁻ is a counterion;    -   X¹ is —O— or —NH—;    -   X² is —O— or —NH—;    -   L is a divalent linking selected from a direct bond or, a lower        alkylene having 1 to 8 carbon atoms, optionally terminated with        or interrupted by one or more divalent connecting groups        selected from sulfonamide (—SO₂NH—), amide —(C═O)N—, ester        —(C═O)—O—, ether —O—, sulfide —S—, sulfone (—SO₂—), phenylene        —C₆H₄—, urethane —NH(C═O)—O—, urea —NH(C═O)NH—, thiourea        —NH(C═S)—NH—, amide —(C═O)NH—, amine —NR— (where R is defined as        alkyl having 1 to 6 carbon atoms) or combinations thereof;    -   Z is either a polymerizable ethylenically unsaturated group        selected from but not limited to methacrylamido-, acrylamido-,        methacryloyl-, acryloyl-, or styryl- or optionally Z is a        reactive functional group, capable of forming a covalent bond        with a polymer or matrix. Such groups include but are not        limited to —Br, —OH, —SH, —CO₂H, and —NH₂. In one embodiment, Z        is

-   -    wherein R is H or CH₃;    -   the bond from the central benzene ring is to the ortho, meta or        para position on the adjacent pyridinium rings; and    -   —B(OH)₂ may be in the ortho, meta or para position.

A specific pyridinium boronic acid quencher, termed P-3, having thestructure below is disclosed in accordance with preferred embodiments ofthe present invention.

A method of making P-3 is disclosed in accordance with anotherembodiment of the present invention comprising the steps of:

Another pyridinium boronic acid quencher having the generic structurebelow is disclosed in accordance with preferred embodiments of thepresent invention.

wherein

-   -   X⁻ is a counterion;    -   X¹ is —O— or —NH—;    -   X² is —O— or —NH—;    -   L is a divalent linking selected from a direct bond or, a lower        alkylene having 1 to 8 carbon atoms, optionally terminated with        or interrupted by one or more divalent connecting groups        selected from sulfonamide (—SO₂NH—), amide —(C═O)N—, ester        —(C═O)—O—, ether —O—, sulfide —S—, sulfone (—SO₂—), phenylene        —C₆H₄—, urethane —NH(C═O)—O—, urea —NH(C═O)NH—, thiourea        —NH(C═S)—NH—, amide —(C═O)NH—, amine —NR— (where R is defined as        alkyl having 1 to 6 carbon atoms) or combinations thereof;    -   Z is either a polymerizable ethylenically unsaturated group        selected from but not limited to methacrylamido-, acrylamido-,        methacryloyl-, acryloyl-, or styryl- or optionally Z is a        reactive functional group, capable of forming a covalent bond        with a polymer or matrix. Such groups include but are not        limited to —Br, —OH, —SH, —CO₂H, and —NH₂. In one embodiment, Z        is

-   -    wherein R is H or CH₃;    -   the ambiguously depicted bonds are in the ortho, meta or para        position; and    -   —B(OH)₂ may be in the ortho, meta or para position.

Another pyridinium boronic acid quencher, termed P-4, having thestructure below is disclosed in accordance with preferred embodiments ofthe present invention.

A method of making P-4 is disclosed in accordance with anotherembodiment of the present invention comprising the steps of:

Some pyridinium quenchers disclosed herein encompass monovalentstructures with a single benzyl boronic acid group. P-1 is one suchrepresentative of this new class of polymerizable, benzyl boronic acidpyridinium salts. It is a simple molecule that is easy to make fromreadily available intermediates and it performs like viologen-basedhydrogels.

Other embodiments of pyridinium quenchers, termed polypyridiniumquenchers have three or more benzyl boronic acid groups (e.g., T-1 andT-2). These compounds differ from both monovalent pyridinium variantssuch as P-1 and from viologens, which comprise two benzyl boronic acidgroups. T-1 and T-2 are fully N-alkylated polypyridines, wherein thepyridine rings are directly coupled (i.e., no linking group betweenrings). They can also be classified as extended conjugation viologens(i.e., two pyridinium rings connected by a conjugated bridging moiety).The T-2 polypyridinium quencher comprises a trifunctional moiety thatlinks to a reactive group and two terpyridinium groups.

As disclosed herein, P2, P3 and P4 are bis-pyridiniums. Boronic acidsubstituted polypyridiniums are another class of preferred quenchers,wherein there are more than two rings coupled together. The term“polypyridinium” includes: a discrete compound comprised of three ormore pyridinium groups covalently bonded together by a linking group, apolymer comprised of pyridinium repeat units in the chain, a polymerwith pyridinium groups pendant to the chain, a dendrimer comprised ofpyridinium units, preferably including pyridinium terminal groups, anoligomer comprised of pyridinium units, preferably including pyridiniumendgroups, and combinations thereof.

In some embodiments, the quenchers disclosed herein are substituted withat least two boronic acid groups and are water-soluble or dispersiblepolymers or hydrogels comprised of polypyridinium boronic acids.Alternatively, the polypyridinium boronic acid is directly bonded to aninert substrate. Quencher precursors comprised of polypyridinium boronicacids include low molecular weight polypyridinium boronic acids furthersubstituted with polymerizable groups or coupling groups.

Some of the structures disclosed herein are quencher precursors (i.e.,monomers) used to make the sensing polymers. It would not be practicalto use the monomers as quenchers for in vitro applications due to theirreactivity. On the other hand, non-polymerizable versions can be madethat are useful for in vitro applications. These embodiments comprise anon-polymerizable group (e.g., the methacrylamido group can be replacedwith a solubilizing group such as a PEG substituent).

In some embodiments, the monovalent- or polypyridinium-boronic acidadduct may be a discrete compound having a molecular weight of about 400daltons or greater. In other embodiments, it may also be a pendant groupor a chain unit of a water-soluble or water-dispersible polymer with amolecular weight greater than about 10,000 daltons. In one embodiment,the quencher-polymer unit may be non-covalently associated with apolymer matrix and is physically immobilized therein. In yet anotherembodiment, the quencher-polymer unit may be immobilized as a complexwith a negatively charge water-soluble polymer.

In other embodiments, the monovalent- or polypyridinium-boronic acidmoiety may be a pendant group or a chain unit in a crosslinked,hydrophilic polymer or hydrogel sufficiently permeable to the analyte(e.g., glucose) to allow equilibrium to be established.

In other embodiments, the quencher may be covalently bonded to a secondwater-insoluble polymer matrix by a linker as described herein. Thequencher may be linked to a water-insoluble polymer matrix at one or twosites in some embodiments.

Analyte Sensors

The chemical indicator systems used in accordance with preferredembodiments of the present invention comprise a fluorophore operablycoupled to an analyte binding moiety, wherein analyte binding causes anapparent optical change in the fluorophore concentration (e.g., emissionintensity). It is further desired that the fluorophore has differentacid and base forms that exhibit a detectable difference in spectralproperties such that ratiometric pH sensing may be enabled; see e.g.,co-pending U.S. patent application Ser. No. 11/671,880. For example, aglucose binding moiety, e.g., P-1 that is operably coupled to afluorescent dye, such as HPTS-triCysMA, will quench the emissionintensity of the fluorescent dye, wherein the extent of quenching isreduced upon glucose binding resulting in an increase in emissionintensity related to glucose concentration. P-1 has at least one boronicacid per pyridinium whereas other pyridinium quenchers may have multiplepyridinium rings, some of which are not substituted with boronic acidgroups.

In further preferred embodiments, the indicator systems also comprise ameans for immobilizing the sensing moieties (e.g., dye-quencher) suchthat they remain physically close enough to one another to react(quenching). Where in vivo sensing is desired, such immobilizing meansare preferably insoluble in an aqueous environment (e.g.,intravascular), permeable to the target analytes, and impermeable to thesensing moieties. Typically, the immobilizing means comprises awater-insoluble organic polymer matrix. For example, the dye andquencher may be effectively immobilized within a DMAA(N,N-dimethylacrylamide) hydrogel matrix, which allows glucose sensingin vivo.

Some exemplary fluorophores and immobilizing means are set forth ingreater detail below. In some embodiments, useful dyes include pyraninederivatives (e.g. hydroxypyrene trisulfonamide derivatives and thelike). In other embodiments, the dye may be one of the polymericderivatives of hydroxypyrene trisulfonic acid.

In one preferred embodiment, the fluorescent dye may be HPTS-TriCys-MA:

Of course, in some embodiments, substitutions other than Cys-MA on theHPTS core are consistent with aspects of the present invention, as longas the substitutions are negatively charged and have a polymerizablegroup. Either L or D stereoisomers of cysteine may be used. In someembodiments, only one or two of the sulfonic acids may be substituted.Likewise, in variations to HPTS-CysMA shown above, other counterionsbesides NBu₄ ⁺ may be used, including positively charged metals, e.g.,Na⁺. In other variations, the sulfonic acid groups may be replaced withe.g., phosphoric, carboxylic, etc. functional groups.

In some embodiments, for use in vitro not involving a moving stream, thesensing components are used as individual (discrete) components. Thefluorophore and quencher are mixed together in liquid solution, analyteis added, the change in fluorescence intensity is measured, and thecomponents are discarded. Polymeric matrices that can be used to trapthe sensing components to prevent leaching need not be present.Optionally, the sensing components are immobilized which allows theiruse to measure analytes in a moving stream.

Applications In Vivo

For in vivo applications, the analyte sensor is used in a moving streamof physiological fluid which contains one or more polyhydroxyl organiccompounds or is implanted in tissue such as muscle which contains saidcompounds. Therefore, it is preferred that none of the sensing moietiesescape from the sensor assembly. Thus, for use in vivo, the sensingcomponents are preferably part of an organic polymer sensing assembly.Soluble dyes and quenchers can be confined by a semipermeable membranethat allows passage of the analyte but blocks passage of the sensingmoieties. This can be realized by using as sensing moieties solublemolecules that are substantially larger than the analyte molecules(molecular weight of at least twice that of the analyte or greater than1000 preferably greater than 5000); and employing a selectivesemipermeable membrane such as a dialysis or an ultrafiltration membranewith a specific molecular weight cutoff between the two so that thesensing moieties are quantitatively retained.

Preferably, the sensing moieties are immobilized in an insoluble polymermatrix, which is freely permeable to glucose. The polymer matrix iscomprised of organic, inorganic or combinations of polymers thereof. Thematrix may be composed of biocompatible materials. Alternatively, thematrix is coated with a second biocompatible polymer that is permeableto the analytes of interest.

The function of the polymer matrix is to hold together and immobilizethe fluorophore and quencher moieties while at the same time allowingcontact with the analyte, and binding of the analyte to the boronicacid. To achieve this effect, the matrix must be insoluble in themedium, and in close association with it by establishing a high surfacearea interface between matrix and analyte solution. For example, anultra-thin film or microporous support matrix is used. Alternatively,the matrix is swellable in the analyte solution, e.g. a hydrogel matrixis used for aqueous systems. In some instances, the sensing polymers arebonded to a surface such as the surface of a light conduit, orimpregnated in a microporous membrane. In all cases, the matrix must notinterfere with transport of the analyte to the binding sites so thatequilibrium can be established between the two phases. Techniques forpreparing ultra-thin films, microporous polymers, microporous sol-gels,and hydrogels are established in the art. All useful matrices aredefined as being analyte permeable.

Hydrogel polymers are used in some embodiments. The term, hydrogel, asused herein refers to a polymer that swells substantially, but does notdissolve in water. Such hydrogels may be linear, branched, or networkpolymers, or polyelectrolyte complexes, with the proviso that theycontain no soluble or leachable fractions. Typically, hydrogel networksare prepared by a crosslinking step, which is performed on water-solublepolymers so that they swell but do not dissolve in aqueous media.Alternatively, the hydrogel polymers are prepared by copolymerizing amixture of hydrophilic and crosslinking monomers to obtain a waterswellable network polymer. Such polymers are formed either by additionor condensation polymerization, or by combination process. In thesecases, the sensing moieties are incorporated into the polymer bycopolymerization using monomeric derivatives in combination withnetwork-forming monomers. Alternatively, reactive moieties are coupledto an already prepared matrix using a post polymerization reaction. Saidsensing moieties are units in the polymer chain or pendant groupsattached to the chain.

The hydrogels useful in this invention are also monolithic polymers,such as a single network to which both dye and quencher are covalentlybonded, or multi-component hydrogels. Multi-component hydrogels includeinterpenetrating networks, polyelectrolyte complexes, and various otherblends of two or more polymers to obtain a water swellable composite,which includes dispersions of a second polymer in a hydrogel matrix andalternating microlayer assemblies.

Monolithic hydrogels are typically formed by free radicalcopolymerization of a mixture of hydrophilic monomers, including but notlimited to HEMA, PEGMA, methacrylic acid, hydroxyethyl acrylate, N-vinylpyrrolidone, acrylamide, N,N′-dimethyl acrylamide, and the like; ionicmonomers include methacryloylaminopropyl trimethylammonium chloride,diallyl dimethyl ammonium chloride, vinyl benzyl trimethyl ammoniumchloride, sodium sulfopropyl methacrylate, and the like; crosslinkersinclude ethylene dimethacrylate, PEGDMA, trimethylolpropane triacrylate,and the like. The ratios of monomers are chosen to optimize networkproperties including permeability, swelling index, and gel strengthusing principles well established in the art. In one embodiment, the dyemoiety is derived from an ethylenically unsaturated derivative of a dyemolecule, such as8-acetoxypyrene-1,3,6-N,N′,N″-tris(methacrylamidopropylsulfonamide), thequencher moiety is derived from an ethylenically unsaturated viologensuch as 4-N-(benzyl-3-boronic acid)-4′-N′-(benzyl-4ethenyl)-dipyridiniumdihalide (m-SBBV) and the matrix is made from HEMA and PEGDMA. Theconcentration of dye is chosen to optimize emission intensity. The ratioof quencher to dye is adjusted to provide sufficient quenching toproduce the desired measurable signal.

In some embodiments, a monolithic hydrogel is formed by a condensationpolymerization. For example, acetoxy pyrene trisulfonyl chloride isreacted with an excess of PEG diamine to obtain a tris-(amino PEG)adduct dissolved in the unreacted diamine. A solution of excesstrimesoyl chloride and an acid acceptor is reacted with4-N-(benzyl-3-boronic acid)-4′-N′-(2 hydroxyethyl) bipyridinium dihalideto obtain an acid chloride functional ester of the viologen. The tworeactive mixtures are brought into contact with each other and allowedto react to form the hydrogel, e.g. by casting a thin film of onemixture and dipping it into the other.

In other embodiments, multi-component hydrogels wherein the dye isincorporated in one component and the quencher in another are preferredfor making the sensor of this invention. Further, these systems areoptionally molecularly imprinted to enhance interaction betweencomponents and to provide selectivity for glucose over other polyhydroxyanalytes. Preferably, the multicomponent system is an interpenetratingpolymer network (IPN) or a semi-interpenetrating polymer network(semi-IPN).

The IPN polymers are typically made by sequential polymerization. First,a network comprising the quencher is formed. The network is then swollenwith a mixture of monomers including the dye monomer and a secondpolymerization is carried out to obtain the IPN hydrogel.

The semi-IPN hydrogel is formed by dissolving a soluble polymercontaining dye moieties in a mixture of monomers including a quenchermonomer and polymerizing the mixture. In some embodiments, the sensingmoieties are immobilized by an insoluble polymer matrix which is freelypermeable to polyhydroxyl compounds. Additional details on hydrogelsystems have been disclosed in US Patent Publications Nos.US2004/0028612, and 2006/0083688 which are hereby incorporated byreference in their entireties.

The polymer matrix is comprised of organic, inorganic or combinations ofpolymers thereof. The matrix may be composed of biocompatible materials.Alternatively, the matrix is coated with a second biocompatible polymerthat is permeable to the analytes of interest. The function of thepolymer matrix is to hold together and immobilize the fluorescent dyeand quencher moieties while at the same time allowing contact with theanalytes (e.g., polyhydroxyl compounds, H⁺ and OH⁻), and binding of thepolyhydroxyl compounds to the boronic acid. Therefore, the matrix isinsoluble in the medium and in close association with it by establishinga high surface area interface between matrix and analyte solution. Thematrix also does not interfere with transport of the analyte to thebinding sites so that equilibrium can be established between the twophases. In one embodiment, an ultra-thin film or microporous supportmatrix may be used. In another embodiment, the matrix that is swellablein the analyte solution (e.g. a hydrogel matrix) can be used for aqueoussystems. In some embodiments, the sensing polymers are bonded to asurface such as the surface of a light conduit, or impregnated in amicroporous membrane. Techniques for preparing ultra-thin films,microporous polymers, microporous sol-gels, and hydrogels have beenestablished in the prior art.

EXAMPLE 1

Compound 27—To a solution of 3,5-dibromopyridine (0.47 g, 2.0 mmol) inanhydrous 1,4-dioxane (15 mL), was added an aqueous solution of K₃PO₄ (2M, 3 mL), followed by PPh₃ (0.21 g, 0.8 mmol) and Pd(OAc)₂ (0.05 g, 0.2mmol). After stirring for 5 min.,[5-(methoxycarbonyl)pyridin-3-yl]boronic acid (0.9 g, 5 mmol) was added,and the reaction was refluxed for 2 h. while a gentle and steady streamof argon was bubbled through the solution. After cooling to roomtemperature, water (10 mL) was added, and the reaction was extractedwith EtOAc (50 mL). The organic layer was separated, dried over MgSO₄,concentrated in vacuo, and purified by flash column chromatography (100%CHCl₃) to give compound 27 (0.3 g, 51%). TLC: R_(f)=0.49 (2%MeOH/CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ 4.01 (s, 3H), 8.08 (t, J=2.1 Hz,1H), 8.49 (t, J=2.1 Hz, 1H), 8.77 (d, J=2.2 Hz, 1H), 8.80 (d, J=2.0 Hz,1H), 9.0 (d, J=2.3 Hz, 1H), 9.28 (d, J=2.0 Hz, 1H).

Compound 28—To a suspension of compound 27 (0.3 g, 1.0 mmol) and3-pyridineboronic acid (0.14 g, 1.1 mmol) in anhydrous 1,4-dioxane (5mL), was added PPh₃ (0.05 g, 0.2 mmol) and Pd(OAc)₂ (0.01 g, 0.05 mmol)followed by an aqueous solution of K₃PO₄ (2 M, 1.1 mL). The reaction wasrefluxed for 1.5 h. while a gentle and steady stream of argon wasbubbled through the solution. After cooling to room temperature, EtOAc(10 mL) was added, and the organic layer was washed with dilute NaHCO₃(5 mL), brine (5 mL), dried over MgSO₄, concentrated in vacuo, andpurified by flash column chromatography (2%-20% methanol in DCM) to givecompound 28 (0.24 g, 83%). ¹H NMR (CDCl₃, 500 MHz) δ 4.02 (s, 3H), 7.48(dd, J=8.1, 4.5 Hz, 1H), 7.97 (dt, J=7.9, 1.9 Hz, 1H), 8.10 (t, J=2.2Hz, 1H), 8.57 (t, J=2.1 Hz, 1H), 8.72 (dd, J=4.8, 4.2 Hz, 1H), 8.93 (m,3H), 9.08 (d, J=2.3 Hz, 1H), 9.30 (d, J=1.9 Hz, 1H).

Compound 29—To a suspension of compound 28 (0.24 g, 0.8 mmol) in THF (15mL), MeOH (10 mL), and water (3 mL), was added LiOH (0.03 g, 1.4 mmol).After stirring for 15 min., the reaction became clear. The reaction wasstirred for 18 h., and the volatiles were then evaporated. The remainingaqueous solution was diluted with NaOH (1 M, 20 mL), washed with DCM (10mL), and acidified to pH 4 with KHSO₄ (1 M) to precipitate the product.The white solid was collected by filtration, washed with water and driedunder vacuum 29 (0.21 g, 95%). ¹H NMR (DMSO-d₆, 500 MHz) δ 7.56 (ddd,J=7.9, 4.8, 0.8 Hz, 1H), 8.33 (ddd, J=7.9, 2.3, 1.7 Hz, 1H), 8.59 (t,J=2.2 Hz, 1H), 8.66 (dd, J=4.8, 1.6 Hz, 1H), 8.68 (t, J=2.2 Hz, 1H),9.04 (dd, J=4.1, 2.2 Hz, 2H), 9.12 (m, 2H), 9.26 (d, J=2.3 Hz, 1H).

Compound 30—To a cooled (0° C.) suspension of compound 29 (0.21 g, 0.76mmol) in dichloromethane (50 mL), was addedN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (0.17 g,0.9 mmol), 1-hydroxy-benzotriazole hydrate (0.12 g, 0.9 mmol), andtriethylamine (0.15 mL, 1.1 mmol). After stirring for 30 min. at 0° C.,N-(3-aminopropyl)methacrylamide hydrochloride (0.16 g, 0.9 mmol) andtriethylamine (0.15 mL, 1.1 mmol) were added. The reaction was stirredfor 18 h, then washed with saturated NaHCO₃ (3×25 mL). The DCM layer wasdried with MgSO₄, reduced in volume in vacuo, and purified by flashcolumn chromatography (2%-20% methanol in DCM) to give compound 30 (0.19g, 62%) as a white solid. TLC: R_(f)=0.50 (10% MeOH/DCM on a platetreated with triethylamine). ¹H NMR (CDCl₃, 500 MHz) δ 1.84 (p, J=6.0Hz, 2H), 1.98 (s, 3H), 3.51 (q, J=6.5 Hz, 2H), 3.55 (q, J=6.1 Hz, 2H),5.39 (s, 1H), 5.79 (s, 1H), 6.30 (t, 1H), 7.46 (dd, J=7.9, 4.9 Hz, 1H),7.98 (ddd, J=7.9, 2.3, 1.7 Hz, 1H), 8.06 (t, 1H), 8.18 (t, J=2.2 Hz,1H), 8.58 (t, J=2.2 Hz, 1H), 8.71 (dd, J=4.8, 1.5 Hz, 1H), 8.92 (d,J=2.1 Hz, 1H), 8.94 (d, J=1.9 Hz, 1H), 8.98 (d, J=2.2 Hz, 1H), 9.04 (d,J=2.2 Hz, 1H), 9.22 (d, J=2.1 Hz, 1H).

Compound T-1—2-Bromomethylphenyl boronic acid (0.6 g, 2.8 mmol) wasadded to a solution of compound 30 (0.19 g, 0.47 mmol) in DMF (3 mL) andethylene glycol (0.16 mL, 2.8 mmol). The reaction was stirred at 55° C.for 72 h. Diethylether (20 mL) was added to separate the product as anoil. The solvent was decanted, and the remaining oil was sonicated inacetone until it became a pale yellow powder. The solid was collected bycentrifugation, washed with acetone several times and dried under argon(0.29 g, 59%). ¹H NMR (D2O, 500 MHz) δ 1.89 (p, J=6.8 Hz, 2H), 2.22 (s,3H), 3.36 (t, J=6.8 Hz, 2H), 3.50 (t, J=6.6 Hz, 2H), 5.40 (s, 1H), 5.65(s, 1H), 6.10 (s, 2H), 6.16 (s, 2H), 6.17 (s, 2H), 7.60 (m, 9H), 7.80(m, 3H), 8.26 (dd, J=8.1, 6.3 Hz, 1H), 8.93 (d, J=8.5 Hz, 1H), 9.07 (t,J=6.2 Hz, 1H), 9.25 (m, 2H), 9.28 (t, J=1.6 Hz, 1H), 9.32 (d, J=5.3 Hz,2H), 9.39 (s, 1H), 9.46 (s, 1H).

EXAMPLE 2

Compound 19—To a suspension of 3,5-dibromopyridine (2.1 g, 9.0 mmol) and3-pyridineboronic acid (1.1 g, 9.0 mmol) in anhydrous 1,4-dioxane (40mL), was added an aqueous solution of K₃PO₄ (2 M, 9 mL), followed byPPh₃ (0.5 g, 2.0 mmol) and Pd(OAc)₂ (0.11 g, 0.5 mmol). The reaction wasrefluxed for 2 h. while a gentle and steady stream of argon was bubbledthrough the solution. After cooling to room temperature, the aqueouslayer was extracted with EtOAc (1×100 mL). The organic layer was washedwith dilute NaHCO₃ (3×50 mL) and brine (1×50 mL), dried over MgSO₄,concentrated in vacuo, and purified by flash column chromatography(2%-20% methanol in DCM) to give compound 19 (1.3 g, 61%). TLC:R_(f)=0.63 (10% MeOH/DCM). ¹H NMR (CD₃OD, 500 MHz) δ 7.59 (ddd, J=8.0,4.9, 0.8 Hz, 1H), 8.18 (ddd, J=8.0, 2.3, 1.6 Hz, 1H), 8.38 (t, J=2.1 Hz,1H), 8.63 (dd, J=4.9, 1.5 Hz, 1H), 8.72 (d, J=2.2 Hz, 1H), 8.85 (d,J=2.0 Hz, 1H), 8.88 (dd, J=2.4, 0.7 Hz, 1H).

Compound 20—A three-necked round-bottomed flask equipped with athermometer was charged with compound 19 (1.2 g, 5.1 mmol), toluene (8mL), THF (3 mL), and triisopropylborate (1.4 mL, 6.0 mmol). Aftercooling to −40° C. (dry ice/acetone), n-butyllithium (1.6 M in hexanes,3.75 mL) was slowly added over the course of 30 min. The reaction wasthen allowed to warm to −20° C., and HCl (2M, 5 mL) was added. When thereaction reached room temperature, the aqueous layer was removed andadjusted to pH 7.6 with NaOH (3M, 2 mL), saturated with NaCl, andextracted with THF (3×6 mL). The THF layers were combined, dried withMgSO₄, evaporated to an oil, diluted with CH₃CN (40 mL), and heated at70° C. for 30 min. The solution was let crystallize at 4° C. for 72 h.The yellow solid was filtered, washed with ice-cold CH₃CN, and air-dried(0.38 g, 37%). ¹H NMR (CD₃OD, 500 MHz) δ 7.61 (dd, J=7.7, 4.9 Hz, 1H),8.21 (dt, J=8.0, 1.9 Hz, 1H), 8.60 (s, 1H), 8.65 (dd, J=4.9, 1.4 Hz,1H), 8.72 (s, 1H), 8.89 (d, J=2.2 Hz, 1H), 8.91 (d, J=1.9 Hz, 1H).

Compound 29 via 31—To a suspension of compound 20 (0.37 g, 1.85 mmol)and ethyl-5-bromonicotinate (0.39 g, 1.68 mmol) in anhydrous 1,4-dioxane(20 mL), was added PPh₃ (0.1 g, 0.37 mmol) and Pd(OAc)₂ (0.02 g, 0.09mmol) followed by an aqueous solution of K₃PO₄ (2 M, 1.65 mL). Thereaction was refluxed for 1.5 h. while a gentle and steady stream ofargon was bubbled through the solution. After cooling to roomtemperature, EtOAc (10 mL) was added, and the organic layer was washedwith water (10 mL), dried over MgSO₄, and evaporated in vacuo to givecrude 31 as a yellow solid. To a suspension of this solid in methanol(20 mL) and water (5 mL), was added LiOH (0.12 g, 5.1 mmol), and thereaction was stirred for 4 h. After removal of methanol in vacuo, morewater was added (10 mL), and the basic aqueous solution was washed withEtOAc (3×5 mL), then adjusted to pH˜4 with KHSO₄ (1 M), which resultedin precipitation. The white precipitate was collected by filtration,washed with acetone and hexanes, and dried to give compound 29 (0.31 g,67%). ¹H NMR (DMSO-d₆, 500 MHz) δ 7.56 (ddd, J=7.9, 4.8, 0.8 Hz, 1H),8.33 (ddd, J=7.9, 2.3, 1.7 Hz, 1H), 8.59 (t, J=2.2 Hz, 1H), 8.66 (dd,J=4.8, 1.6 Hz, 1H), 8.68 (t, J=2.2 Hz, 1H), 9.04 (dd, J=4.1, 2.2 Hz,2H), 9.12 (m, 2H), 9.26 (d, J=2.3 Hz, 1H).

Compound 32—To a cooled (0° C.) suspension of compound 29 (0.3 g, 1.0mmol) in dichloromethane (15 mL), was addedN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (0.23 g,1.2 mmol), 1-hydroxy-benzotriazole hydrate (0.16 g, 1.2 mmol), andtriethylamine (0.28 mL, 2.0 mmol). After stirring for 30 min. at 0° C.,compound 10 (0.14 g, 0.4 mmol) was added. The reaction was stirred for24 h. White precipitate formed. After the addition of saturated NaHCO₃(50 mL), a significant amount of solid remained in both layers. Thesolid was filtered, washed with DCM, NaHCO₃, hexanes, and dried to givecompound 32 (0.19 g, 61%). ¹H NMR (CD₃OD, 500 MHz) δ 1.60 (m, 2H), 1.73(m, 4H), 1.86 (s, 3H), 1.96 (m, 2H), 3.23 (m, 4H), 3.49 (m, 2H), 4.58(dd, J=9.2, 5.1 Hz, 2H), 5.29 (s, 1H), 5.63 (s, 1H), 7.60 (dd, J=7.6,5.0 Hz, 2H), 8.26 (m, 2H), 8.46 (s, 1H), 8.49 (s, 1H), 8.53 (s, 1H),8.63 (d, J=4.6 Hz, 2H), 8.72 (s, 1H), 8.93 (m, 7H), 9.05 (s, 3H).

Compound T-2—2-Bromomethylphenyl boronic acid (0.47 g, 2.2 mmol) wasadded to a solution of compound 32 (0.19 g, 0.24 mmol) in DMF (4 mL) andethylene glycol (0.12 mL, 2.2 mmol). The reaction was stirred at 55° C.for 72 h. Acetone (40 mL) was added, and the resulting precipitate wassonicated until a fine pink powder was obtained. The solid was collectedby centrifugation, washed with acetone several times and dried underargon (0.30 g, 60%). ¹H NMR (D₂O, 500 MHz) δ 1.53 (m, 2H), 1.70 (m, 4H),1.78 (s, 3H), 1.95 (m, 2H), 3.20 (m, 4H), 3.47 (m, 2H), 4.46 (t, 1H),5.30 (s, 1H), 5.53 (s, 1H), 6.09 (s, 4H), 6.14 (s, 4H), 6.16 (s, 4H),7.56 (m, 18H), 7.77 (m, 6H), 8.25 (t, 2H), 8.91 (d, J=7.9, 2H), 9.10 (d,2H), 9.26 (m, 6H), 9.33 (m, 4H), 9.38 (m, 2H), 9.46 (s, 1H), 9.50 (s,1H).

EXAMPLE 3

Compound 33—To a cooled (0° C.) suspension of isonicotinic acid (0.57 g,4.7 mmol) in dichloromethane (80 mL), was addedN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.1 g, 5.6mmol), 1-hydroxy-benzotriazole hydrate (0.76 g, 5.6 mmol), andtriethylamine (0.8 mL, 5.6 mmol). After stirring for 30 min. at 0° C.,N-(3-aminopropyl)methacrylamide hydrochloride (1.0 g, 5.6 mmol) andtriethylamine (0.8 mL, 5.6 mmol) were added. The reaction was stirredfor 2 h., then washed with saturated NaHCO₃ (3×25 mL). The DCM layer wasdried with MgSO₄, reduced in volume in vacuo, and purified by flashcolumn chromatography (2%-10% methanol in DCM) to give compound 33 (0.37g, 32%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 1.77 (m, 2H), 2.01(s, 3H), 3.47 (q, J=6.5 Hz, 2H), 3.50 (q, J=6.1 Hz, 2H), 5.40 (s, 1H),5.80 (s, 1H), 6.36 (t, 1H), 7.76 (d, J=6.1 Hz, 2H), 7.90 (t, 1H), 8.76(d, J=6.1 Hz, 1H).

Compound P-1—2-Bromomethylphenyl boronic acid (0.45 g, 2.1 mmol) wasadded to a solution of compound 33 (0.19 g, 0.24 mmol) in acetonitrile(75 mL). The reaction was stirred at 50° C. for 16 h. The reaction wasconcentrated in vacuo until 5 mL remained. Ether (20 mL) was added, andthe precipitate was sonicated, and collected by centrifugation. Toremove excess starting material, the white precipitate was sonicated inDCM for 1 h. The DCM was decanted from the oil, and the oil was thensonicated in ether until a white powder was obtained. The solid wascollected by centrifugation, washed with ether several times and driedunder argon (0.23 g, 36%). ¹H NMR (D₂O, 500 MHz) δ 1.87 (m, 2H), 1.89(s, 3H), 3.34 (t, J=6.7 Hz, 2H), 3.48 (t, J=6.7 Hz, 2H), 5.40 (s, 1H),5.65 (s, 1H), 6.03 (s, 2H), 7.55 (m, 3H), 7.76 (d, J=7.1 Hz, 1H), 8.25(d, J=6.3 Hz, 2H), 8.96 (d, J=6.5 Hz, 1H).

EXAMPLE 4

Compound 10—To a cooled (0° C.) solution of N,N-di-boc-lysine(dicyclohexylammonium) salt (4.2 g, 8.0 mmol) in dichloromethane (200mL), was added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (1.8 g, 9.6 mmol), 1-hydroxy-benzotriazole hydrate (1.3 g,9.6 mmol), and triethylamine (1.3 mL, 9.6 mmol). After stirring for 30min. at 0° C., N-(3-aminopropyl)methacrylamide hydrochloride (1.7 g, 9.6mmol) and triethylamine (1.3 mL, 9.6 mmol) were added. The reaction wasstirred for 8 h., then washed with saturated NaHCO₃ (3×75 mL). The DCMlayer was dried with MgSO₄, reduced in volume in vacuo, and purified byflash column chromatography (2%-20% methanol in DCM) to give compound 6.TLC: R_(f)=0.71 (10% MeOH/DCM). The appropriate fractions were pooledand concentrated to about 5 mL (not taken to dryness to avoidpolymerization), then 1.25 M methanolic HCl (30 mL) was added and thereaction was stirred for 48 h., and concentrated in vacuo to give 10 asa white foam (2.1 g, 78%). ¹H NMR (D₂O, 500 MHz) δ 1.44 (p, J=8.3 Hz,2H), 1.71 (p, J=7.8 Hz, 2H), 1.78 (p, J=6.9 Hz, 2H), 1.90 (m, 2H), 1.92(s, 3H), 3.00 (t, J=7.7 Hz, 2H), 3.29 (m, 4H), 3.95 (t, J=6.7 Hz, 1H),5.44 (s, 1H), 5.67 (s, 1H).

Compound 34—Compound 10 (0.4 g, 1.2 mmol) was suspended in a solution ofdichloromethane (50 mL) and triethylamine (0.9 mL, 6.4 mmol) and cooledto 0° C. Then, isonicotinic acid (0.4 g, 3.2 mmol),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (0.73 g,3.8 mmol), 1-hydroxy-benzotriazole hydrate (0.51 g, 3.8 mmol), andtriethylamine (0.9 mL, 6.4 mmol) were added. After stirring for 1 h. at0° C., the reaction was sonicated for 3 h. to help dissolve the solids.After washing with saturated NaHCO₃ (3×75 mL), the DCM layer was driedwith MgSO₄, reduced in volume in vacuo, and purified by flash columnchromatography (2%-20% methanol in DCM, silica gel pretreated with 1%triethylamine) to give compound 34 (19 mg, 3%). TLC: R_(f)=0.59 (15%MeOH/DCM, plate pretreated with triethylamine).

Compound P-2—2-Bromomethylphenyl boronic acid (0.02 g, 0.1 mmol) wasadded to a solution of compound 34 (18.7 mg, 40 μmol) in DMF (1 mL). Thereaction was stirred at 55° C. for 72 h. Diethylether (20 mL) was addedto separate the product as an oil. The solvent was decanted, and theremaining oil was sonicated in ether until it became a beige powder. Thesolid was collected by centrifugation, washed with ether several timesand dried under argon (35 mg, 96%). ¹H NMR (D₂O, 500 MHz) δ 1.46 (m,2H), 1.69 (m, 4H), 1.86 (s, 3H), 1.90 (m, 2H), 3.22 (m, 4H), 3.44 (t,J=6.8 Hz, 2H), 4.42 (t, J=7.4 Hz, 1H), 5.38 (s, 1H), 5.61 (s, 1H), 6.02(s, 2H), 6.03 (s, 2H), 7.56 (m, 6H), 7.76 (d, J=7.6 Hz, 2H), 8.23 (d,J=6.7 Hz, 2H), 8.28 (d, J=6.7 Hz, 2H), 8.97 (m, 4H).

EXAMPLE 5

EXAMPLE 6

EXAMPLE 7

Sensor Preparation and Testing

The quencher, P-1, was dissolved in 41.4 μL of a stock solutioncontaining N,N′-dimethylacrylamide (100 mg) andN,N′-methylenebismethacrylamide (2 mg). This quencher solution (20.7 μL)was then added to a solution containing HPTS-TriCys-MA (50 μL of a 2 mMaqueous solution), HCl (20 μL of a 100 mM solution),2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (10 μL, of a40 mg/mL solution), and DI water (99.3 μL). Some of this solution wasthen polymerized onto the tip of a fiber optic sensor by heating at 37°C. for 24 h. to form a hydrogel.

The sensor was tested by placing it in solutions containing differentglucose concentrations ranging from 0 mg/dL to 400 mg/dL. The hydrogelindicator chemistry at the tip of the optical fiber was excited withlight at a wavelength of 470 nm. Fluorescence emission was monitoredbetween 520-700 nm. The results are illustrated in FIG. 1.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications, and publications, referredto above, are hereby incorporated by reference.

1. The reactive compound:

wherein: X— is a counterion; X¹ is —O— or —NH—; X² is —O— or —NH—; L isa divalent linking group comprising a direct bond or a lower alkylenehaving 1 to 8 carbon atoms, optionally terminated with or interrupted byone or more divalent connecting groups selected from the groupconsisting of sulfonamide (—SO₂NH—), amide —(C═O)N—, ester —(C═O)—O—,ether —O—, sulfide —S—, sulfone (—SO₂—), phenylene —C₆H₄—, urethane—NH(C═O)—O—, urea —NH(C═O)NH—, thiourea —NH(C═S)—NH—, amide —(C═O)NH—,and amine —NR—, where R is defined as alkyl having 1 to 6 carbon atoms;and Z is either a reactive, ethylenically unsaturated group or areactive functional group, capable of forming a covalent bond with apolymer or matrix.
 2. The compound of claim 1, wherein Z is a reactive,ethylenically unsaturated group selected from the group consisting ofmethacrylamido-, acrylamido-, methacryloyl-, acryloyl- and styryl-. 3.The compound of claim 1, wherein Z is a reactive functional groupcapable of forming a covalent bond with a polymer or matrix selectedfrom the group consisting of —Br, —OH, —SH, —CO₂H, and —NH₂.
 4. Thecompound of claim 1, wherein Z is

wherein R is H or CH₃.
 5. The reactive compound of claim 1, wherein saidcompound is:


6. A method of making the compound of claim 5, comprising the steps of:


7. The compound:

wherein: Z is a reactive, ethylenically unsaturated group selected fromthe group consisting of methacrylamido-, acrylamido-, methacryloyl-,acryloyl- and styryl-, or wherein Z is a reactive functional groupcapable of forming a covalent bond with a polymer or matrix; Y is atrivalent connecting group selected from

 where R is H or a lower alkyl, and

X¹ and X² are —O— or —NH—; and L¹, L², and L³ are selected from a directbond or, a lower alkylene having 1 to 8 carbon atoms, optionallyterminated with or interrupted by one or more divalent connecting groupsselected from sulfonamide (—SO₂NH—), amide —(C═O)N—, ester —(C═O)—O—,ether —O—, sulfide —S—, sulfone (—SO₂—), phenylene —C₆H₄—, urethane—NH(C═O)—O—, urea —NH(C═O)NH—, thiourea —NH(C═S)—NH—, amide —(C═O)NH—,amine —NR—(where R is defined as alkyl having 1 to 6 carbon atoms) orcombinations thereof.
 8. The compound of claim 7, wherein Z is apolymerizable, ethylenically unsaturated group selected from the groupconsisting of methacrylamido-, acrylamido-, methacryloyl-, acryloyl- andstyryl-.
 9. The compound of claim 7, wherein Z is a reactive functionalgroup capable of forming a covalent bond with a polymer or matrixselected from the group consisting of —Br, —OH, —SH, —CO₂H, and —NH₂.10. The compound:

wherein n is an integer from 1-10.
 11. A method of making the compoundof claim 10, comprising the steps of:


12. The compound:

wherein: X⁻ is a counterion; X¹ is —O— or —NH—; X² is —O— or —NH—; L isa divalent linking selected from a direct bond or, a lower alkylenehaving 1 to 8 carbon atoms, optionally terminated with or interrupted byone or more divalent connecting groups selected from sulfonamide(—SO₂NH—), amide —(C═O)N—, ester —(C═O)—O—, ether —O—, sulfide —S—,sulfone (—SO₂—), phenylene —C₆H₄—, urethane —NH(C═O)—O—, urea—NH(C═O)NH—, thiourea —NH(C═S)—NH—, amide —(C═O)NH—, amine —NR—(where Ris defined as alkyl having 1 to 6 carbon atoms) or combinations thereof;Z is a reactive group selected from a coupling group or an olefinicallyunsaturated group, or Z is

 wherein R is H or CH₃; the bond from the central benzene ring is to theortho, meta or para position on the adjacent pyridinium rings; and—B(OH)₂ may be in the ortho, meta or para position.
 13. The compound:


14. A method of making the compound of claim 13, comprising the stepsof:


15. The compound:

wherein X⁻ is a counterion; X¹ is —O— or —NH—; X² is —O— or —NH—; L is adivalent linker selected from the group consisting of a direct bond anda lower alkylene having 1 to 8 carbon atoms, optionally terminated withor interrupted by one or more divalent connecting groups selected fromthe group consisting of sulfonamide (—SO₂NH—), amide —(C═O)N—, ester—(C═O)—O—, ether —O—, sulfide —S—, sulfone (—SO₂—), phenylene —C₆H₄—,urethane —NH(C═O)—O—, urea —NH(C═O)NH—, thiourea —NH(C═S)—NH—, amide—(C═O)NH—, amine —NR—(where R is defined as alkyl having 1 to 6 carbonatoms) or combinations thereof; Z is either a polymerizableethylenically unsaturated group selected from the group consisting ofmethacrylamido-, acrylamido-, methacryloyl-, acryloyl-, or styryl- oroptionally Z is a reactive functional group capable of forming acovalent bond with a polymer or matrix; the ambiguously depicted bondsare in the ortho, meta or para position; and —B(OH)₂ may be in theortho, meta or para position.
 16. The compound of claim 15, wherein Z is

wherein R is H or CH₃.
 17. The compound of claim 15, wherein Z isselected from the group consisting of —Br, —OH, —SH, —CO₂H, and —NH₂.18. The compound:


19. A method of making the compound of claim 18, comprising the stepsof:


20. A glucose sensor comprising one or more of the compounds of any oneof the claims 1, 4, 5, 7, 10, 12, 13, and 15-18, and a fluorescent dye.21. The glucose sensor of claim 20, wherein said one or more of thecompounds are in the form of a polymer.
 22. The glucose sensor of claim20, further comprising a glucose permeable immobilizing means, e.g., apolymer matrix or a semipermeable membrane.